They selectively transport mono- or disaccharides across plasma or intracellular membranes, and are involved in a number of essential physiological processes... The functions of SWEETs are best characterized in plants... The structure of TySemiSWEET was determined by molecular replacement using the recently reported structure of a SemiSWEET protein from L. biflexa (LbSemiSWEET) as search model and refined to 2.4 Å resolution (Supplementary information, Figure S1 and Table S1)... Six TySemiSWEET molecules that are arranged into three dimers were found in each asymmetric unit (Figure 1A and Supplementary information, Figure S1)... Interestingly, when TM1 and TM2 in each 3-helix bundle are aligned on the same plane, the position of TM3 in SemiSWEETs is on the opposite side to that of the MFS 3-helix bundle (Supplementary information, Figure S3)... The bulky side groups of the two Phe41 in LbSemiSWEET dimer close the central pocket in the midway of the membrane, whereas Met47 residues in TySemiSWEET leave enough space for an elongated central pocket (Figure 1E, 1F and Supplementary information, Figure S4)... Structural comparison of TySemiSWEET and LbSemiSWEET provides important clue to understanding substrate selectivity of SemiSWEETs... If the central cavities observed in TySemiSWEET and LbSemiSWEET represent the primary binding site for ligands in SemiSWEETs, two questions immediately stand out... The positions of the cavity in TySemiSWEET and LbSemiSWEET, both located closer to the periplasmic side, deviate from those in the known structures of sugar transporters, in which a primary substrate binding site is usually placed in the midway of the membrane... An inward-open structure is yet to be captured to elucidate the alternating access cycle of SemiSWEETs... On top of that, an intriguing and critical question in the study of SemiSWEETs and SWEETs is the driving force for their conformational changes... It remains to be characterized whether SemiSWEETs and SWEETs are facilitative uniporters or secondary active co-transporters... The structures reported here and previously lay out the foundation to address these important questions... The atomic coordinates have been deposited in the Protein Data Bank with the accession code 4RNG.

fig1: Crystal structure of the SemiSWEET from T. yellowstonii (TySemiSWEET) in an occluded conformation. (A) Overall structure of the dimeric TySemiSWEET. The two protomers are colored green and cyan. (B-D) The dimer interface of TySemiSWEET consists of three clusters of H-bonds between residues on TM1 of one protomer and TM2 of the other, including a pair of H-bonds at the extracellular side (B), a pair close to the center of the membrane (C), and an extensive H-bond network on the cytoplasmic side (D). The H-bonds, together with extensive van der Waals contacts between the two protomers, sealed the dimer in an occluded conformation. (E-F) The central cavity of TySemiSWEET is considerably larger than that of LbSemiSWEET. Residues Phe41 from the two protomers of LbSemiSWEET close the central pocket at approximately the midway of the membrane, whereas the corresponding Met47 residues in TySemiSWEET leave enough space for an elongated cavity. A sucrose molecule can be accommodated by TySemiSWEET, but not LbSemiSWEET (right panels). All structure figures were prepared with PyMol10.

Mentions:
Six TySemiSWEET molecules that are arranged into three dimers were found in each asymmetric unit (Figure 1A and Supplementary information, Figure S1). While two dimers are arranged in a parallel fashion, the third one is positioned in the opposite orientation, further supporting the dimeric assembly of SemiSWEETs6. Within each dimer, the two parallel protomers that exhibit almost identical conformations are related by 180° rotation around an axis perpendicular to the membrane plane. Within each protomer, TM1 and TM2 are connected by an extended linker (L1-2), and TM3 is positioned between TM1 and TM2 (Figure 1A). Note that the L1-2 linker is enriched with positively charged residues (Supplementary information, Figure S2). According to the “positive-inside rule” and the topological analysis of AtSWEET11, the L1-2 should be located on the cytoplasmic side, which leaves the N-terminus of each protomer on the periplasmic side of the membrane (Figure 1A)6,7.

fig1: Crystal structure of the SemiSWEET from T. yellowstonii (TySemiSWEET) in an occluded conformation. (A) Overall structure of the dimeric TySemiSWEET. The two protomers are colored green and cyan. (B-D) The dimer interface of TySemiSWEET consists of three clusters of H-bonds between residues on TM1 of one protomer and TM2 of the other, including a pair of H-bonds at the extracellular side (B), a pair close to the center of the membrane (C), and an extensive H-bond network on the cytoplasmic side (D). The H-bonds, together with extensive van der Waals contacts between the two protomers, sealed the dimer in an occluded conformation. (E-F) The central cavity of TySemiSWEET is considerably larger than that of LbSemiSWEET. Residues Phe41 from the two protomers of LbSemiSWEET close the central pocket at approximately the midway of the membrane, whereas the corresponding Met47 residues in TySemiSWEET leave enough space for an elongated cavity. A sucrose molecule can be accommodated by TySemiSWEET, but not LbSemiSWEET (right panels). All structure figures were prepared with PyMol10.

Mentions:
Six TySemiSWEET molecules that are arranged into three dimers were found in each asymmetric unit (Figure 1A and Supplementary information, Figure S1). While two dimers are arranged in a parallel fashion, the third one is positioned in the opposite orientation, further supporting the dimeric assembly of SemiSWEETs6. Within each dimer, the two parallel protomers that exhibit almost identical conformations are related by 180° rotation around an axis perpendicular to the membrane plane. Within each protomer, TM1 and TM2 are connected by an extended linker (L1-2), and TM3 is positioned between TM1 and TM2 (Figure 1A). Note that the L1-2 linker is enriched with positively charged residues (Supplementary information, Figure S2). According to the “positive-inside rule” and the topological analysis of AtSWEET11, the L1-2 should be located on the cytoplasmic side, which leaves the N-terminus of each protomer on the periplasmic side of the membrane (Figure 1A)6,7.

They selectively transport mono- or disaccharides across plasma or intracellular membranes, and are involved in a number of essential physiological processes... The functions of SWEETs are best characterized in plants... The structure of TySemiSWEET was determined by molecular replacement using the recently reported structure of a SemiSWEET protein from L. biflexa (LbSemiSWEET) as search model and refined to 2.4 Å resolution (Supplementary information, Figure S1 and Table S1)... Six TySemiSWEET molecules that are arranged into three dimers were found in each asymmetric unit (Figure 1A and Supplementary information, Figure S1)... Interestingly, when TM1 and TM2 in each 3-helix bundle are aligned on the same plane, the position of TM3 in SemiSWEETs is on the opposite side to that of the MFS 3-helix bundle (Supplementary information, Figure S3)... The bulky side groups of the two Phe41 in LbSemiSWEET dimer close the central pocket in the midway of the membrane, whereas Met47 residues in TySemiSWEET leave enough space for an elongated central pocket (Figure 1E, 1F and Supplementary information, Figure S4)... Structural comparison of TySemiSWEET and LbSemiSWEET provides important clue to understanding substrate selectivity of SemiSWEETs... If the central cavities observed in TySemiSWEET and LbSemiSWEET represent the primary binding site for ligands in SemiSWEETs, two questions immediately stand out... The positions of the cavity in TySemiSWEET and LbSemiSWEET, both located closer to the periplasmic side, deviate from those in the known structures of sugar transporters, in which a primary substrate binding site is usually placed in the midway of the membrane... An inward-open structure is yet to be captured to elucidate the alternating access cycle of SemiSWEETs... On top of that, an intriguing and critical question in the study of SemiSWEETs and SWEETs is the driving force for their conformational changes... It remains to be characterized whether SemiSWEETs and SWEETs are facilitative uniporters or secondary active co-transporters... The structures reported here and previously lay out the foundation to address these important questions... The atomic coordinates have been deposited in the Protein Data Bank with the accession code 4RNG.